羊口疮病毒B2L、F1L基因融合真核表达质粒诱导小鼠的免疫应答及IL-2对其作用影响的研究

为评价羊口疮病毒(OrfV)B2L、F1L基因融合真核表达质粒pVAX1-B2L-F1L免疫小鼠诱导的体液和细胞免疫应答及IL-2对其免疫作用的影响。本研究将构建的pVAX1-B2L-F1L真核质粒转染MDBK细胞后,采用RT-PCR和间接免疫荧光试验(IFA)检测B2L-F1L融合基因在MDBK细胞中的表达;将pVAX1-B2L-F1L、pVAX1-B2L-F1L+pVAX1-IL-2、pVAX1空载体、生理盐水对照组KM系小鼠通过后腿肌肉注射的方式免疫,采用ELISA方法检测免疫小鼠血清中OrfV特异性抗体以及Th1型(IL-2、IFN-γ)、Th2型(IL-4、IL-6)细胞因子;MTT法检测小鼠脾淋巴细胞增殖反应。结果显示,pVAX1-B2L-F1L重组质粒能够在MDBK细胞中表达;pVAX1-B2L-F1L+pVAX1-IL-2联合免疫组小鼠血清抗体及IL-2和IFN-γ水平均显著高于pVAX1-B2L-F1L组;该联合免疫组小鼠血清IL-4、IL-6细胞因子水平与pVAX1-B2L-F1L组相比差异不显著(p>0.05);该联合免疫组小鼠的脾淋巴细胞增殖水平高于pVAX1-B2L-F1L组(p<0.05)。由此表明,pVAX1-B2L-F1L能够诱导小鼠产生OrfV特异性体液免疫和细胞免疫应答,联合pVAX1-IL-2诱导的免疫反应以细胞免疫应答为主,且能够促进Th1型细胞因子的分泌。本研究为OrfV基因工程疫苗的研制提供了参考依据。

Evaluation of the immune response induced by OrfV B2L-F1L fusion gene eukaryotic expression plasmid alone or combined with Interleukin-2 gene in mice

The aim of this study was to evaluate the effect of OrfV F1L and B2L fusion gene eukaryotic expression plasmid pVAX1-B2L-F1L alone or combined with IL-2 on mice humoral and cellular immune responses. In this study, pVAX1-B2L-F1L eukaryotic plasmid was transfected into MDBK cells, and the expression of B2L-F1L fusion gene in MDBK cells was detected by RT-PCR and indirect immunofluorescence assay(IFA). The constructed pVAX1-B2L-F1L, pVAX1-B2L-F1L+pVAX1-IL-2, pVAX1 empty carrier and normal saline control KM-line mice were immunized by hind leg intramuscular injection of the mice. Specific OrfV serum antibody, Th1 type cytokines(IL-2, IFN-γ) and Th2 type cytokines(IL-4, IL-6) were detected by ELISA. MTT assay was used to detect lymphocyte proliferation in mice. The results showed that pVAX1-B2L-F1L recombinant plasmid was highly expressed in MDBK cells. The amount of anti-OrfV antibodies, cytokines of IL-2 and IFN-γ in pVAX1-B2L-F1L + pVAX1-IL-2 combined immunization group significantly higher than those in pVAX1-B2L-F1L group. There were no significant differences of IL-4 and IL-6 cytokine level in serum between pVAX1-B2L-F1L group and pVAX1-IL-2 group(p>0.05). The spleen lymphocyte proliferation level of pVAX1-B2L-F1L + pVAX1-IL-2 group was higher than that of pVAX1-B2L-F1L group(p<0.05). Therefore,pVAX1-B2L-F1L can highly induce OrfV-specific humoral and cellular immunities in mice when it was combined with pVAX1-IL-2. The immune response induced by pVAX1-IL-2 is mainly cellular immune response, promoting the secretion of Th1 cytokines. The rsults provide reference for the development of OrfV genetic engineering vaccine.